1 alpha,25-Dihydroxyvitamin D3 exerts rapid nongenomic effects on rat osteoblast-like cells independent of the classic nuclear receptor. These effects include changes in phospholipid metabolism and cell calcium. Intracellular calcium itself has been proposed to regulate intracellular pH in osteoblast cell lines. The purpose of this study was to determine the effect of 1 alpha,25-dihydroxyvitamin D3 on intracellular pH, the relationship of changes in calcium to changes in pH, and the role of pH changes in genomic activation. 1 alpha,25-Dihydroxyvitamin D3 increased intracellular pH within 10 min in rat osteoblast-like cells, an effect that was inhibited by removal of extracellular sodium and by the biologically inactive epimer 1 beta,25-dihydroxyvitamin D3. The hormone increased intracellular calcium in Quin 2 loaded cells in the presence and absence of extracellular sodium. The 1 alpha,25-dihydroxyvitamin D3-induced increments in osteocalcin and osteopontin mRNA levels were abolished in sodium-free medium. The results indicate that 1 alpha,25-dihydroxyvitamin D3-induced increments in cellular calcium precede cell alkalinization and that these changes in intracellular pH may modulate steady-state mRNA levels of genes induced by vitamin D.